Stereospecific microbiological hydrolysis process

ABSTRACT

The stereospecific microbiological hydrolysis of racemic 2substituted-(optionally Delta 1) 3-acyloxy-5oxocyclopentaneheptanoic acids and their lower alkyl esters with Saccharomyces sp. N.R.R.L. Y-7342 affords the corresponding 3 Alpha -hydroxy acids or the corresponding esters. Those compounds are intermediates in the preparation of optically active prostaglandin derivatives which are useful as pharmacological agents.

United States Patent 1 Colton et a1.

[ Apr. 2, 1974 0 STEREOSPECIFIC MICROBIOLOGICAL HYDROLYSIS PROCESS [75]Inventors: Frank B. Colton, Evanstom william J. Marsheck, Lake Zurich;Masateru Miyano, Morton Grove, all of I11.

[73] Assignee: G. D. Searle & Co., Chicago, 111. [22] Filed: Apr. 14,1972' i [21] Appl. No.: 244,293

[52] US. Cl 195/30, 195/51 R, 260/473,

7 260/476, 260/514, 260/520 [51] Int. Cl..; Cl2d 1/02 [58] Field ofSearch 195/30, 51 R [56] References Cited UNITED STATES PATENTS3,290,226 12/1966 Bealet al ..l9 5/30 Primary Examiner-Alvin E.Tenenholtz Attorney, Agent, or Firm-John M. Brown 57 ABSTRACT- Thestereospecific microbiological hydrolysis of racemic'2-substituted-(optionally A 3-acyloXy-5- oxocyclopentaneheptanoic acidsand their lower alkyl 5 Claims, No Drawings STEREOSPECIFICMICROBIOLOGICAL HYDROLYSIS PROCESS The present invention is concernedwith the stereospecific microbiological hydrolysis of substituted 3-acyloxycyclopentaneheptanoic acids and esters. In particular, it isconcerned with the stereospecific microbiological hydrolysis of racemic2-substituted 3-acyloxy- 5-oxocyclopentaneheptanoic acids and esters ofthe formula JLH.

. wherein R is a hydrogen or lower alkyl radical, R is -a styryl,formyl, a,BB-dihydroxyphen ethyl, 3-hydroXy-1- octenyl or3-oxo-l-octenyl radical, R is an acyl radical containing 1 to 7 carbonatoms and the dotted line represents an optional 1,2-double bond bycontacting the above compounds with Saccharomyces sp. N.R.R.L. Y-7342,thus forming the 3a-hydroxy compounds while leaving the 3B-acetoxycompounds unreacted.

The lower alkyl radicals represented by the above formula areillustrated by methyl ethyl, propyl, pentyl,

hexyl, heptyl and the branched-chain isomers thereof,

the acyl radicals represented contain up to 7 carbon atoms and areillustrated by acetyl, propionyl and isobutyryl and the wavy linerepresents the, racemic mixture of a and B isomers.

It has been surprisingly discovered that, when Saccharomyces sp.N.R.R.L. (Northern Regional Research Laboratory, 1815 North UniversityStreet, Peoria, lll. 61604 Y-7342 is fermented with racemic2-substituted-(optionally A) 3-acyloxy-5-oxo =cyclopentaneheptanoicacids or their lower alkyl esters, hydrolysis selectively occurs toafford the corresponding l-3a-hydroxy acid or ester, while the 3,8-acyloxy isomer remains unreacted. The products of this process may berecovered by usual extraction or chromatographic methods such as thosedescribed in the examples.

Starting materials useful for the practice of the instant process aresuitably manufactured from styrylglyoxal, conveniently prepared by theselenous acid oxidation of 4-phenyl-3-buten-2-one, and the dialkylesters of 3-oxoundecane-l,l l-dioic acid. Dimethyl 3-oxoundecane-l,ll-dioate is thus saponified with potassium hydroxide and the resultingdicarboxylic acid is allowed to react with styrylglyoxal, thus affordingd1- l4-phenyl-9, l 2-dioxo-l l-hydroxytetradec- 1 3-enoic acid.Cyclization of the latter intermediate in the presence of potassiumhydroxide results in dl-'3-hydroxy'-5- styryl compounds. A convenientreagent is osmium tet roxide. For example, dl-methyl 3-acetoxy-5-oxo-2-styrylcyclopent-l-eneheptanoate is thus contacted at room temperaturewith osmium tetroxide in dioxane to produce methyl3-acetoxy-5-oxo-2-(a,B- dihydroxyphenethyl )-cyclopentl -eneheptanoate.

A convenient procedure for manufacture of the 2- formyl compoundsconsists of cleaving the glycol'structure of the corresponding2-(a,/3-dihydroxyphenethyl) substances. d l -Methyl3-acetoxy-5-oxo-2-(a,B-dihydroxyphenethyl)cyclopentl-eneheptanoate inethanol is contacted with aqueous sodium periodate, thus affording dl-methyl 3- acetoxy-Z-formyl-S-oxocyclopentl -eneheptanoate. The2-formyl compounds are alternatively produced from the corresponding2-styryl derivatives by combining the 'hydroxylation and cleavageprocesses. dl- Methyl 3-hydroxy-5 -oxo-2-styrylcyclopentl eneheptanoatein aqueous dioxane is thus allowed to react with osmium tetroxide andsodium periodate to afford dl-methyl2-formyl-3-hydroxy-5-oxocyclopentl-eneheptanoate.

Reduction of the A starting materials used in the instant processprovides the corresponding cyclopentaneheptanoic acids and esters.Typical reducing agents are chromous sulfate or zinc metal. As anexample of the reduction process, d1-2-formyl-3-acetoxy-5-oxocyclopent-l-eneheptanoic acid, when treated with zinc powder inaqueous acetic acid affords d1-2-formyl-3-acetoxy-5-oxocyclopentaneheptanoic acid.

The compounds produced by the instant process are useful asintermediates in the manufacture of optically active prostanoic acidderivatives of the following structural formula 0 I cnmcoon on W whereinR is hydrogen or a lower alkyl radical, as defined hereinbefore, Z is acarbonyl or hydroxymethylene group and the dotted line indicates anoptional 8,.12 double bond.

A specific example of the manufacture of one of the latter compounds isthe reaction of methyl Ba-hydroxy-2-formyl-5-oxocyclopent-l-eneheptanoate with hexanoylmethylene triphenylphosphorane to afford methyl 1 la-hydroxy-9, l 5-dioxoprosta-8( 12), l3- ,dienoate. These prostanoic acid derivatives display valuablepharmacological properties. They are thus hypotensive, smoothmuscle-contracting, anti-bacterial, anti-protozoal, anti-fungal andpepsin-inhibitory agents.

In the practice of this invention, the substrate may be added to thegrowing culture or it may be added to the nutrient medium beforeincubation with the microorganism. The culture medium should containusable sources of carbon and nitrogen and an adequate sterile airsupply. Typically, air can be supplied by exposing a large surface areaof the culture medium to air or by passing air through the submergedculture. Suitable nitrogen sources are illustrated by soybean meal, cornsteep liquor, mean extracts, peptone, and/or distillers solubles orother useful organic materials. Synthetics such as nitrates and ammoniumcompounds may also be used.

Sources of carbon suitable for use in the process of this inventioninclude meat extracts, peptone and similar materials. Additionallysuitable compounds are glycerol, glucose, fructose, dextrose, sucrose,lactose, maltose, dextrins, starch and whey. These materials may be usedeither in purified states or as concentrates such as whey concentrate,corn steep liquor, grain mashes and the like, or as mixtures of theabove.

It is often desirable to add an anti-foaming agent to the culturemedium. Also, sources of phosphate, magnesium and/or ferrous ions may beincorporated in the culture medium as growth promoting agents. Buffersmay also be added to control pH at desired levels.

Process operating conditions may be varied and are not critical. Howeverthey should notbe so severe as to retard the fermentation or destroy theorganism. Typically, concentrations of substrate in the range of 001-50percent, temperatures in the range of 2535C. and reaction times of l-lOdays are common but not limiting.

The invention will appear more fully from the examples which follow.However, those examples are not to be considered as limiting theinvention either in spirit or in scope as many variations both inmaterials and methods will be apparent to one skilled in the art.Ternperatures are given in degrees Centigrade (C.) and quantities ofmaterial in parts by weight unless otherwise specified.

EXAMPLE 1 A solution containing 100 parts of 4-phenyl-3-buten- 2-one,106 parts of selenous acid, 160 parts of dioxane and 20 parts of wateris heated to the reflux temperature. After the initial vigorous reactionhas subsided,

boiling at about 120 at 2.5 mm. pressure.

EXAMPLE 2 A solution of 38.2 parts of dimethyl 3-oxoundecane- 1,1l-dioate in 200 parts by volume of percent aqueous potassium hydroxideis stored at O5 for about 3 days, then is adjusted to pH 5 by theaddition of concentrated aqueous citric acid. To that mixture is added asolution which is prepared by heating 21.9 parts of styrylglyoxal in 50parts by volume of 50 percent aqueous methanol at 6575 for about 20minutes, then adding 60 parts of methanol. To the resulting reactionmixture is added 30 parts by volume of l M pH 4.5-5.0 citrate buffer andstirring-at room temperature is continued for about 3 hours, duringwhich time carbon dioxide gas is evolved. The precipitated product iscollected by filtration, thus affording the half potassium salt ofdl-l4-phenyl-9,l2-dioxo-l l-hydroxytetradeclS-enoic acid, melting atabout 105. Further purification by recrystallization from methanolaffords the pure compound, melting at about lO7.5.

The latter half potassium salt is dissolved in water and the resultingaqueous solution is acidified by the addition of dilute hydrochloricacid. The resulting acidic mixture is extracted with ether and the etherlayer is separated, washed with water, dried over anhydrous sodiumsulfate and concentrated to dryness. The resulting solid resiude ispurified by recrystallization from chloroform-ether to yieldd1-l4-phenyl-9,l2- dioxo-l l-hydroxytetradec-l3-enoic acid, melting atabout 81.5-83.

EXAMPLE 3 To 3,000 parts by volume of an aqueous solution containing 6.7parts of potassium hydroxide is added, with stirring at 2l23 over aperiod of about 2% hours, a solution of 10.4 parts of d1-14-phenyl-9,l2-dioxo-l l-hydroxytetradec-l3-enoic acid in 187 parts of chloroform.After completion of the addition, the reaction mixture is stirred for anadditional 2 hours, then is made acidic by adding 10 parts of oxalicacid dihydrate. The acidic mixture is extracted with chloroform and theorganic layer is washed with dilute aqueous sodium chloride, then driedover anhydrous sodium sulfate and concentrated to dryness under reducedpressure. The resulting residue is recrystallized first from benzene,then from chloroform-ether to yield d1-3-hydroxy-5-oxo-2-styrylcyclopentr l-eneheptanoic acid, which displays amelting point at about 1 18. This compound displays an ultravioletabsorption maximum at about 325 millimicrons with a molecular extinctioncoefficient of about 36,400.

EXAMPLE 4 A mixture containing 44.3 parts of dl 3-hydroxy-5-oxo-2-styrylcyclopent-l-eneheptanoic acid, 11.3 parts of diazomethaneand 700 parts of ether is kept at room temperature for about 5 minutes,at the end of which time acetic acid is-added in order to destroy theexcess reagent. The resulting mixture is then washed with aqeous sodiumbicarbonate, dried over anhydrous sodium sulfate and stripped of solventby distillation under reduced pressure. The residue is purified bychromatography, first on silica gel followed by elution with 50 percentethyl acetate in benzene, then by dry.chromatography on silica gelcontaining 8 percent water, also using 50 percent ethyl acetate inbenzene, thus affording d1 -methyl 3-hydroxy-5-oxo-2-styrylcyclopent- 1eneheptanoate. This compound is characterized by infrared absorptionmaxima, in chloroform, at about 2.75, 2.87, 5.76, 5.88 and 6. l 7microns and by an ultraviolet absorption maximum at about 325millimicrons with a molecular extinction coefficient of about 25,000.

EXAMPLE 5 A solution containing 0.9 part of dl-methyl 3-hydroxy-S-oxo-2-styrylcyclopentl-eneheptanoate, 10 parts of pyridine and2 parts of acetic anhydride is kept at room temperature for about 16hours, then is poured slowly into water. The resulting aqueous mixtureis extracted with ether and the ether layer is separated, washedsuccessively with dilute aqueous sodium bicarbonate and dilute aqueoussodium chloride, then dried over anhydrous sodium sulfate andconcentrated to dryness under reduced pressure. The resulting residue ispurified either by preparative thin layer chromatography using 20percent ethyl acetate in benzene on silica gel or by dry chromatographyon silica gel containing 8 percent water, also using 20 percent ethylacetate in benzene. The resulting product, obtained as an oil, is d 1-methyl 3-acetoxy-5-oxo-2-styrylcyclopentl eneheptanoate. ln chloroform,this compound exhibits infrared absorption maxima at about 5.75, 5.86,6.15 and 8.02 microns. It exhibits also an ultraviolet absorptionmaximum at about 325 millimicrons with a molecular extinctioncoefficient of about 32,700.

EXAMPLE 6 To a solution of 123 parts of d1 methyl 3-acetoxy-5-oxo-Z-styrylcyclopent-l-eneheptanoate in 20 parts of dioxaneis added asolution of 0.81 part of osmium tetroxide in 3.85 parts of dioxane. Theresulting reaction mixture is allowed to stand at room temperature forabout 70 hours, at the end of which time the excess reagent isdecomposed by the addition-of hydrogen sulfide. The resulting solutionis filtered through silica gel containing 8 percent of water and theadsorbent is washed with an ethyl acetate-methanol solution. The

EXAMPLE 7 To a solution of 0.2 part of dl-methyl 3-acetoxy-5-oxo-2-(a,/3-dihydroxyphenethyl)cyclopent-leneheptanoate in 8 parts ofethanol is added a solution of 0.12 part of sodium periodate in 2 partsof water. The resulting reaction mixture is allowed to stand at roomtemperature for about 45 minutes, then is diluted with water andextracted with ether. The ether layer is separated, washed with water,dried over anhydrous sodium sulfate and concentrated to dryness underreduced pressure. The resulting residue is heated under reduced pressurefor about 10 minutes in order-to remove benzaldehyde, thus affording theoily product, which is dl-methyl 3-acetoxy-2-formyl-5-oxocyclopent-l-eneheptanoate. It displays infrared absorption maxima, inchloroform, at about 5.78 and 5.92 microns.

EXAMPLE 8 To a suspension of 214 parts of triphenyl methyl phosphoniumbromide with 1,400 parts of ether, under nitrogen, 'is added, at 0-5,190 parts by volumeof a hexane solution containing 41.9 parts of n-butyllithium. The resulting reaction mixture is allowed to warm to roomtemperature, then is stirred for about 1 hour and cooled to 05. Asolution of 100 parts of nhexanoyl chloride in 700 parts of ether isadded under nitrogen and the resulting mixture is kept at roomtemperature for about 16 hours. At the end of that reaction period theether solution is decanted and washed with dilute hydrobromic acid. Theacidic washing is then shaken with the precipitate and the resultingsolution is extracted with chloroform. The chloroform extract is washedsuccessively with hydrobromic acid and water, dried over anhydroussodium sulfate, concentrated to a small volume and diluted with hexane.The resulting crystals of starting material are removed by filtrationand the filtrate is dissolved in chloroform, then washed successivelywith 20 percent aqueous potassium hydroxide, water, hydrobromic acid andwater, dried over anhydrous sodium sulfate and concentrated to a smallvolume under reduced pressure. Dilution of the resulting solution withcyclohexane results in precipitation of the crystalline product, whichis purified by recrystallization from aqueous ethanol to affordtransparent needle-like crsytals of triphenyl 2-oxoheptyl phosphoniumbromide, melting at about 195.

EXAMPLE 9 A solution of 0. 19 part of triphenyl 2-oxoheptyl phosphoniumbromide in parts of chloroform is shaken with dilute aqueous potassiumhydroxide, then washed with dilute aqueous sodium chloride, dried overanhydrous sodium sulfate, concentrated and dried at room temperatureunder reduced pressure. The resulting residue consisting of 0.16 part ofhexanoylmethylene triphenyl phosphorane is combined with 0.13 part ofdlmethyl 3-acetoxy-2-formyl-5-oxocyclopentl eneheptanoate and dissolvedin 13.2 parts of benzene. The resulting reaction mixture is heated atthe reflux temperature for about 24 hours, then is cooled and strippedof solvent under reduced pressure. The resulting residue is purified bydry column chromatography on silica gel containing 8 percent of water,using 20 percent ethyl acetate in benzene, to afford dl-methyl ll-acetoxy-9,1 5-dioxoprosta-8( 12), 13-dienoate. This compound exhibitsinfrared absorption maxima, in chloroform, at about 5.78 and 6.28microns and mu]- traviolet absorption maximum at about 288.5millimicrons with a molecular extinction coefficient of about 31,300.

EXAMPLE 10 To a solution of 12 parts of dl-l l-acetoxy-9, 15-dioxoprosta-8( l2), 13-dienoic acid in 28 parts of ethanol, cooled toO5, is added dropwise a solution of 3 parts of triethylamine in 275parts of water. To that mixture is added dropwise wth cooling andstirring a solution of 0.32 part of sodium borohydride in 32 parts ofwater. Stirring at approximately 10 is continued for about 25 minutes,at the end of which time the reaction mixture is poured carefully intoexcess aqueous citric acid. Ex-

traction with ether affords an-organic solution, which is washed withwater, dried over anhydrous sodium sulfate and concentrated underreduced pressure to afford d l -1l-acetoxy-l5-hydroxy-9,l5-oxoprosta-8(12),]3- dienoic acid.

EXAMPLE 11 ume of saturated aqueous sodium chloride. Extraction of thatmixture with ether affords an organic solution, which is washed withsaturated aqueous sodium chloride, then dried over anhydrous sodiumsulfate and stripped of solvent under reduced pressure. The resultingresidue is combined with n-hexanoylmethylene triphenyl phosphorane,prepared from 27.2 parts of nhexanoylmethyl triphenyl phosphoniumchloride according to the procedure of Example 9, then is dissolved in amixture of parts of dioxane and 440 parts of benzene. The resultingmixtute is heated under nitrogen at the reflux temperature for about 5/2 hours,

EXAMPLE 12 A medium consisting of parts of commercial pancreatic digestof casein, 5 parts of commercial peptic digest of animal tissue, 20parts of dextrose, and 1,000 parts of distilled water is placed in equalamounts in five 1 liter flasks. The medium is sterilized by heating inan autoclave to 120 at p.s.i. for minutes. To the resulting sterilemedium, then cooled to is added 2 parts of fluid culture ofSaccharomyces sp. N.R.R.L. Y-7342. The flasks are placed on a rotaryshaker at 170 rpm and 1 inch orbit at 28, and the microorganism ispermitted to grow under these conditions for about 48 hours. After thattime a solution of 0.2- part of dl-3-acetoxy-5 oxo-2-styrylcyclopent-1-eneheptanoic acid in 1 part of acetone is added to each flask. Thefermentation is continued with heavy aeration at 230 rpm for 68 hours.The culture, now at a pH of 3.2, is extracted with methylene chlorideand the solvent is removed under reduced pressure. The crudefermentation product which remains is dissolved in ether and extractedwith 5% sodium bicarbonate solution. The bicarbonate extracts areacidified with citric acid, extracted with ether, and the etherealextract is dried over anhydrous sodium sulfate and concentrated todryness. Recrystallization from ethyl acetate-benzene yields purel-3a-hydroxy-5-oxo-2-styrylcyclopent-1- eneheptanoic acid, melting atabout 1 l5and displaying an optical rotation in methanol of about l6.7.

The ethereal mother liquors are concentrated, diluted with n-pentane andextracted with 5 percent sodium bicarbonate. Then the bicarbonateextracts are washed with a mixture of n-pentane and methanol in a ratioof 2.5 to 1, respectively. Acidification of the bicarbonate solutionwith citric acid and extraction with ether, followed by drying of theethereal extract over anhydrous sodium sulfate and concentration todryness affords an oily residue, which, when chromatographed on silicicacid and eluted with benzene containing increasing amounts of ethylacetate, affords 1-3B- acetoxy-S-oxo2-styrylcyclopent-l-eneheptanoicacid. That compound displays an optical rotation in methanol of about21.1.

EXAMPLE 13 A medium consisting of 25 parts of commercial pancreaticdigest of casein, 25 parts of commercial peptic digest of animal tissue,100 parts of dextrose, 5 parts of silicone anti-foam emulsion and 5000parts of distilled water is sterilized in a 7.5 liter stainless steelfermentor by heating in an autoclave to 120C. with 15 p.s.i. steam for50 minutes. To the resulting sterile medium, then cooled to 25, is added250 parts of a fluid culture of Saccharomyces sp. N.R.R.L. Y-7342. Themixture is agitated mechanically at 200 rpm while introducing sterileair at a rate of about 4,000 parts by volume per minute. Themicroorganism is permitted to grow under these conditions for about 20hours at a temperature of about 30. Then a solution of 1 part ofdl-3-acetoxy-5- 0xo-2-styrylcyclopent-l-eneheptanoic acid dissolved in10 parts of acetone is added to the culture and the fermentation isallowed to continue for about 76 hours. The culture, at a pH of about3.1, then is extracted with methylene chloride and the solvent removedunder reduced pressure. The crude fermentation product is dissolved in500 parts by volume of the upper phase of a mixture obtained by shakingtogether 1500 parts by volume of benzene, 500 parts by volume ofmethanol and 200 parts by volume of distilled water and then extractedwith the lower phase of that mixture. The lower phase extractions arecombined and concentrated to yield, after seeding in ethylacetate-benzene, l-3ahydroxy-S-oxo-2-styrylcyclopent l -eneheptanoicacid. That product is identical to the product obtained in Example 12. a

The mother liquors are then chromatographed on silicic acid and elutedusing-benzene containing increasing amounts of ethyl acetate to yieldsuccessively B-(2-hydroxyethyl)-indole and 1- 3B-acetoxy-5-oxo-2-styrylcyclopent- 1 -eneheptanoic acid.

EXAMPLE 14 A medium consisting of- 1,000 parts of commercial pancreaticdigest of casein, 200 parts of commercial enzymatic digest of proteins,200 parts of commercial autolyzed yeast extract, 4000 parts of dextrose,parts of hydrochloric acid, 20 parts of silicone antifoam emulsion and200,000 parts of tap water is mixed in a stainless steel fermentor andsterilized by the addition of steam under pressure to a temperature ofand a final volume of about 250,000 parts by volume. To the resultingsterile medium,-when .cooled to 28, is added 2,000 parts of a fluidculture of Saccharomyces sp. N.R.R.L. Y-7342. That mixture is agitatedmechanically at 200 rpm while introducing sterile air at a rate of about50,000 parts by volume per minute and the microorganism is permitted togrow at these conditions for about 25 hours at a temperature of 28.After this time, a solution of 49 parts ofd1-3-acetoxy-5-oxo-2-styrylcyclopentl-eneheptanoic acid and 200 parts ofacetone is added and the fermentation-is allowed to continue for 30hours. Then the culture, at a pH of 3.8, is extracted with methylenechloride and the solvent is removed under reduced pressure. The crudefermentation product remaining is purified according to the procedurepreviously described in Example 12 to yield pure l-3o-hydroxy-5-oxo-2-styryl=cyc1opent-l-eneheptanoic acid. That product is identical to theproduct of Example 12.

EXAMPLE 15 A medium consisting of 1,000 parts of commercial pancreaticdigest of casein, 200 parts of commercial enzymatic digest of protein,200 parts of commercial autolyzed yeast extract, 4000 parts of dextrose,100 parts of hydrochloric acid, 50 parts of silicone antifoam emulsionand 200,000 parts of tap water is mixed in a stainless steel fermentorwith live steam under pressure to a temperature of l20and a final volumeof about 250,000 parts. To the resulting sterile medium, after coolingto 28, is added 1000 parts of a fluid culture of Saccharomyces sp.N.R.R.L. Y-7342. The mixture is agitated mechanically at 200 rpm. whileintroclucing sterile air at a rate of 60,000 parts by volume per minute.The microorganism is permitted to grow at these conditions for about 17hours at a temperature of about 28. Then a solution of 44 parts ofdl-3-acetoxy- 5 -oxo-2-styrylcyclopentl eneheptanoic acid dissolved in200 parts of acetone is added. The bioconversion is allowed to continuefor about 53 hours, after which time the culture is extracted withmethylene chloride and the solvent removed under reduced pressure. Thecrude fermentation product remaining is purified according to theprocedure described in Example 12 to yield pure l3a-hydroxy-5oxo-2-styrylcyclopent-1- eneheptanoic acid, which compoundis identical to the product of Example 12.

EXAMPLE 16 dine. That mixture is allowed to stand for about 16 hours atroom temperature and then it is poured into a cold solution containing40 parts of d-tartaric acid in 1350 parts of water. That mixture isextracted with ethyl acetate and the organic extracts are washed withwater, dried over anhydrous sodium sulfate and concentrated to dryness.The oily residue which remains is dissolved in benzene andchromatographed on silicic acid. The initial fraction obtained uponelution with percent ethyl acetate-85 percent benzene is recrystallizedfrom benzene-hexane to give colorless crystals of3B-((-)-0-methylmandeloxy)-5oxo-2-styrylcyclopent -1-eneheptanoic acid,melting at about l22124 and displaying an optical rotation in methanolof about 22.2. That compound is further characterized, in chloroform, byabsorption maxima in the infrared spectrum at about 1750, 1710 and 1630reciprocal centimeters and an ultraviolet absorption band, in methanol,

' at about 326 millimicrons with a molecular extinction coefficient ofabout 36,000. The latter fraction, obtained upon elution with 15 percentethyl acetate-85 percent benzene, is recrystallized from benzene-hexaneto give colorless needles of 3a-((-)-0-methylmandeloxy)-5oxo-2-styrylcyclopentl eneheptanoic acid, melting atabout 96-98 and displaying an optical rotation of about 84.2 inmethanol. That compound absorbs in the infrared spectrum, in chloroform,at about 1750, 1710 and 1630 reciprocal'centimeters and has anabsorption band in the ultraviolet spectrum at about 326 millimicronswith a molecular extinction coefficient of about 35,000 in methanol.

EXAMPLE 17 A medium consisting of 25 parts of commercial digest ofcasein, 5 parts of commercial soybean peptone, 4 parts of dextrose, 8parts of sodium chloride, 4 parts of dipotassium phosphate and 1500parts of distilled water is divided into 5 equal portions and placed infive 1 liter flasks. The medium is sterilized by heating in an autoclaveto 120 at 15 p.s.i. for 20 minutes. To the resulting sterile medium,cooled to 25, then is added 0.5 part of a fluid culture ofFlavobacterium dehydrogenans A.T.C.C. 13930. The flasks are incubated ona rotary shaker at 26 for about 24 hours, and, after that time, 0.4 partof 3B-( ()-0-methylmandeloxy-5oxo-2- styrylcyclopent-leneheptanoic aciddissolved in 1 part of acetone is added to each flask. The reaction isallowed to continue for hours with vigorous agitation. The culture isadjusted to pH 4 with citric acid and extracted with methylene chloride,then concentrated to dryness under reduced pressure. The crude productis purified by column chromatography on silicic acid using 50 percentethyl acetate-benzene as eluant. Crystallization of the crude productgives colorless crystals of d-3B-hydroxy-5-oxo-2-styrylcyclopentleneheptanoic acid, melting at about l13114, and displaying an opticalrotation in methanol of about +l6.

EXAMPLE 1 8 A medium consisting of 8 parts of commercial soy protein, 4parts of commercial enzymatic digest of casein, 8 parts of dextrose, 2parts of dipotassium phosphate, and 1500 parts of distilled water isdivided into 5 equal portions in 1 liter flasks. The medium issterilized by heating in an autoclave to at 15 p.s.i. for 20 minutes. Tothe resulting sterile medium, cooled to 25, is added 1 part of a fluidculture of Streptomyces hydrogenans A.T.C.C 19631. Then the flasks areincubated on a rotary shaker at 26 for 6 hours, and 0.3 part of3a-(()-O-methylmandeloxy)-5-oxo-2-styrylcyclopent- 1 eneheptanoic aciddissolved in 1 part of acetone is added to each flask. Thebiotransformation is allowed to continue for about 119 hours withvigorous agitation, and after that time, the culture is adjusted to pH 4with citric acid and extracted with methylene chloride. The crudeproduct is purified by column chromatography on silicic acid using 50percent ethyl acetate-benzene as eluant to yield pure, colorlessl-3ahydroxy-Soxo-2-styrylcyclopent-l-eneheptanoic acid, melting at about112-113 and displaying an optical rotation in methanol of about l5.5.

EXAMPLE 19 When an equivalent quantity of dl-3-acetoxy-2-formyl-S-oxocyclopent-l-eneheptanoic acid is substituted in theprocedure of Example 12, there is produced l'-2-formyl-'3a-hydroxy-S-oxocyclopent-1- eneheptanoic acid. acid.

EXAMPLE 20 By substituting an equivalent quantity of d1-3-acetoxy-2-(oz, fl-dihydroxyphenethyl)-5-oxocyclopentl-eneheptanoic acidin the procedure of Example 12, there is obtained 1-2-(11,,B-dihydroxyphenethyl)-3ahydroxy-S-oxocyclopentl-eneheptanoic acid.

EXAMPLE 21 Substitution of an equivalent quantity of d1-3- acetoxy-2-(3-hydroxyl octenyl)-5-oxocyclopentl eneheptanoic acid in the procedureof Example 12 affords 1-3a-hydroxy-2-(3-hydroxy-1-octenyl)-5-oxocyclopent- 1 eneheptanoic acid.

EXAMPLE 22 By substituting an equivalent quantity of dl-methyl-3acetoxy-Soxo-2-styrylcyclopent-l-eneheptanoate in the procedure ofExample 12 and otherwise following the procedure of Example 12, there isproduced 1- methyl 3a-hydroxy-5 -oxo-2-styrylcyclopentl eneheptanoate.

EXAMPLE 23 When an equivalent quantity of dl-3-acetoxy-2-formyl-S-oxocyclopentaneheptanoic acid is treated according to theprocedure of Example 12, there is afforded l-2-formyl-3a-hydroxy-5-oxocyclopentaneheptanoic acid.

EXAMPLE 24 Substitution of an equivalent quantity of dl-3-acetoxy-2-(3-hydroxy-1-octenyl)-5- oxocyclopentaneheptanoic acid in theprocedure of Example 12 affords 1-3a-hydroxy-2-(3-hydroxy-1- Yoctenyl)-5-oxocyclopentaneheptanoic acid.

EXAMPLE 25 To a solution of 5 parts of dl-3-hydroxy-5-oxo-2-styrylcyclopent-l-eneheptanoic acid in 49 parts of pyridine is added23.2 parts of propionic anhydride and the mixture is allowed to standfor 5 days. After that time, the reaction mixture is poured into 400parts of icewater. After the excess propionic anhydride is decomposed,50 parts by volume of concentrated hydrochloric acid is added and theacidified solution is extracted with ether. The ethereal extract iswashed with an aqueous 2 percent hydrochloric acid solution, then withan aqueous 1 percent sodium chloride solution until neutral, and driedover anhydrous sodium sulfate. The solvent is removed under reducedpressure to afford an oily product. That crude product ischromatographed on silica gel, elution being with 95:5 chloroform-aceticacid, to yield crystalline d1-3- propionyloxy-S-oxo-2-styrylcyclopentl-eneheptanoic acid, melting at about 7173. That material ischaracterized further by an absorption band in the ultraviolet spectrumat about 324 millimicrons with a molecular extinction coefficient ofabout 33,000, absorption in the infrared spectrum at about 1738, 1709,1629 and 1377 reciprocal centimeters and absorption maxima in thenuclear magnetic resonance spectrum at 60 megal-lertz indeuteriochloroform at 8 (ppm) of about 7.05, 6.23, 2.97 and 1.17.

EXAMPLE 26 When an equivalent quantity of isobutyryl anhydride issubstituted in the procedure of Example 25, there is obtainedd1-3-isobutyryloxy-5-oxo-2-styrylcyclopentl-eneheptanoic acid. Thatmaterial melts at about 7980 and is further characterized by absorptionin the ultraviolet spectrum at about 325 millimicrons with a molecularextinction coefficient of about 33,000, absorption in the infraredspectrum at 1729, 1705, 1624, 1372 and l 150 reciprocal centimeters andabsorption maxima in the nuclear magnetic resonance spectrum at 60megal-lertz in deuteriochloroform at 8 (ppm) of about 7.04,

EXAMPLE 27 By substituting an equivalent quantity of dl-3-propionyloxy-S-oxo-2-styrylcyclopentl -eneheptanoic acid in theprocedure of Example 12, there is affordedl-3a-hydroxy-5-oxo-2-styrylcyclopentl-eneheptanoic acid. That product isidentical to the product obtained in Example 12.

EXAMPLE 28 Substitution of an equivalent quantity of dl-3-isobutyryloxy-S-oxo-2-styry1cyclopentl -eneheptanoic acid in theprocedure of Example 12 affords 1-3ahydroxy-5-oxo-2styrylcyclopentl-eneheptanoic acid, identical to the product of Example 12.

What is claimed is: v

1. The process for the production of a compound of the formula 0 cameoOR wherein R is hydrogen or a lower alkyl radical, R is a styryl,a,B-dihydroxyphenethyl, formyl, 3-oxo-loctenyl or 3-hydroxy-l-octenylradical and the dotted line represents an optional 1,2 double bond,which comprises -fermenting a compound of the formula 0 I came 0 0R 5 0Rwherein R, R and the dotted line are as defined above, R is an acylradical containing 1 to 7 carbon atoms and the wavy line represents theracemic mixture, with Saccharomyces sp. N.R.R.L. Y-7342.

2. As in claim 1, the process for the production of a compound of theformula with Saccharomyces sp. N.R.R.L. Y-7342 wherein R, R and R" aredefined as in claim 1.

3. As in claim 1, the process for the production of a compound of theformula (CHDeCOOR 13 14 which comprises fermenting a compound of theforand R" are defined as in claim 1. mula my 4. As in claim 1, theprocess for the production of l- 3a-hydroxy-5-oxo-2-styrylcyclopentl-eneheptanoic (0H,),CO0R acid which comprises fermentingdl-3-acetoxy-5-oxo- 5 2-styrylcyclopentl -eneheptanoic acid withSaccharomyces sp. N.R.R.L. Y-7342.

5. As in claim 1, the process for the production of l-3a-hydroxy-5-oxo-2-styrylcyclopentl -eneheptanoic 0R" acid whichcomprises fermenting dl-3-propionyloxy-5- oxo-2-styrylcyclopent- 1-eneheptanoic acid with Saccharomyces sp. N.R.R.L. Y-7342.

with Saccharomyces sp. N.R.R.L. Y-7342 wherein R

2. As in claim 1, the process for the production of a compound of theformula
 3. As in claim 1, the process for the production of a compoundof the formula
 4. As in claim 1, the process for the production of 1-3Alpha -hydroxy-5-oxo-2-styrylcyclopent-1-eneheptanoic acid whichcomprises fermenting d1-3-acetoxy-5-oxo-2-styrylcyclopent-1-eneheptanoicacid with Saccharomyces sp. N.R.R.L. Y-7342.
 5. As in claim 1, theprocess for the production of 1-3 Alpha-hydroxy-5-oxo-2-styrylcyclopent-1-eneheptanoic acid which comprisesfermenting d1-3-propionyloxy-5-oxo-2-styrylcyclopent-1-eneheptanoic acidwith Saccharomyces sp. N.R.R.L. Y-7342.